Pharmaceutical compositions

ABSTRACT

The present invention relates to pharmaceutical compostions of (1S,4R)-cis-4-[2-amino-6-cyclopropylamino}-9H-purin-9-yl]-2-cyclopentene-1-methanol (1592U89).

This application is filed pursuant to 35 U.S.C. §371 as a United StatesNational Phase Application of International Application No.PCT/EP99/00663 filed Feb. 4, 1999, which claims priority fromGB9802472.2 filed Feb. 6, 1998.

The present invention relates to novel pharmaceutical compositions of(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol.

(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanoland its antiviral use, particularly against HIV infections is describedin European Patent Specification Number 0434450. The succinate salt of(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolis described in WO96/06844. The hemisulfate salt of(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolis described in WO98/52949.

(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol(also known as 1592U89) is currently under clinical investigation as ananti-HIV pharmaceutical agent. There exists a need for the compound tobe prepared in solution form, for example for pediatric use.

Solutions of(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolare bitter-tasting and therefore require the addition of sweeteners andtaste-maskers. However, formulation of(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolin solution is difficult because materials containing a —COOH grouppresent problems with(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolcompatibility. For example, glucose forms an adduct with the compound byreplacing the methanol group on the cyclopentyl ring of(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol.(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhas been found to be incompatible with sucrose (which degrades toglucose and fructose) as well as glucose.

Solutions of(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolcontaining sorbitol result in a color change and the formation of darkcolored particulates at pH 4.5-6.5.

In addition, propylene glycol concentration appears to have an influenceon color formation, with higher levels of propylene glycol (10%) causingcolor formation.

We have found that sorbitol or saccharin or a combination of sorbitoland saccharin are compatible with(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanoland do not lead to the formation of adducts with it. In addition,lowering the pH to about 4.0 eliminates the color change and theformation of particulates. We have also found that combinations offructose, acesulfame and saccharin are compatible with(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol.In addition, we have found that abacavir is stable in pH ranges of about2.0 to about 4.5 and about 6.6 to 7.5. Advantageously, the pH may be 3.8to 4.5. We have also found that the addition of a metal chelator, forexample citrate, improves the stability of abacavir in solution.

According to a first aspect of the invention there is provided apharmaceutical composition comprising(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanoland sorbitol at a range of pH from about 2.0 to about 4.5,advantageously pH 4.0. In an alternative embodiment, there is provided apharmaceutical composition comprising(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanoland saccharin at a range of pH from about 2.0 to about 4.5,advantageously pH 4.1. The pharmaceutical composition may comprise bothsorbitol and saccharin at a range of pH from about 2.0 to about 4.5,advantageously pH 4.1.

A further aspect of the invention includes compositions comprising(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanoland sorbitol and/or saccharin at a pH range of about 6.6 to about 7.5,advantageously pH 7.0. Compositions at about pH 7 may include propyleneglycol or other suitable solubilizer to improve solubility.

According to another aspect of the invention there is provided apharmaceutical composition comprising(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanoland a sweetening agent compatible with said(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolat a pH range of about 2.0 to about 4.5.

In a further aspect of the invention there is provided a pharmaceuticalcomposition comprising(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanoland fructose, acesulfame, and saccharin at a range of pH from about 2.0to about 4.5, advantageously pH 4.0. Alternatively, the compositions maybe formulated at a pH range of about 6.6 to 7.5, advantageously pH 7.0.

According to a further aspect of the present invention there is provideda pharmaceutical composition comprising(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,sorbitol, saccharin and citrate at a range of pH from about 2.0 to about4.5. The citrate ion concentration may advantageously be in the range ofabout 0.01M to about 0.13M. Advantageously, sodium citrate and citricacid may be used.

The compositions of the present invention may also comprise one or morepharmaceutically acceptable organic solvents, for example, propyleneglycol, polypropylene glycol, polyethylene glycol, and the like;pharmaceutically acceptable alcohols, for example ethanol or2-(2-ethoxyethoxy)ethanol and the like; antioxidants, for example,edetate disodium, malic acid, fumaric acid, or sodium metabisulfite, andthe like; pharmaceutically acceptable acids, for example, hydrochloricacid, acetic acid, citric acid, sulfuric acid, and the like; and oils orsurfactants, and the like.

The compositions of the present invention may also comprise otherpharmaceutically acceptable sweetening agents and/or flavoring agents,for example, aspartame, sucralose, and the like and/or cherry flavor,artificial banana flavor, caramel, chocolate mint flavor, grape flavor,wild cherry flavor, raspberry flavor, strawberry flavor, citrus flavor,orange flavor, pineapple flavor, citrus lime flavor, citrus creamflavor, cherry vanilla flavor, creme de menthe flavor and the like.

According to the present invention, any ester of hydroxybenzoate(parabens) or combination of such esters may be used, including methyland propyl paraben and butyl and propyl paraben combinations. Sodiumbenzoate (0.02-0.5% w/v) or potassium sorbate (0.05-0.2% w/v) may beused as preservatives.

In a further aspect of the present invention, there is provided(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolcompositions containing methyl paraben and propyl paraben. For oralsolutions and suspensions, the range of methyl paraben concentration maybe 0.15-0.2% (1.5 mg/mL to 2 mg/mL) and the range of propyl parabenconcentration may be 0.01% to 0.02% (0.1 to 0.2 mg/mL).

According to a further aspect of the present invention, any suitablebuffer may be used to provide a pH>5.5. Advantageously, sodium citrateor phosphate may be used. To achieve a pH range of about 2.0 to about4.5, advantageously citrate, fumarate, glutarate, malate, maleate,tartrate or acetate may be used.

Included in the invention are the pharmaceutically acceptable salts,esters, or salts of such esters of(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,or any other compound which, upon administration of a safe andtherapeutically effective amount of the compound to a human subject, iscapable of providing (directly or indirectly) the antivirally activemetabolite or residue thereof.

Preferred esters in accordance with the invention are independentlyselected from the following group: (1) carboxylic acid esters in whichthe non-carbonyl moiety of the carboxylic acid portion of the estergrouping is selected from straight or branched chain alkyl (for example,methyl, n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (forexample, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl(for example, phenoxymethyl), aryl (for example, phenyl optionallysubstituted by, for example, halogen, C₁₋₄ alkyl, or C₁₋₄ alkoxy), oramino; (2) sulphonate esters, such as alkyl- or aralkylsulphonyl (forexample, methanesulphonyl); (3) amino acid esters (for example, L-valylor L-isoleucyl); and (4) phosphonate esters. In such esters, unlessotherwise specified, any alkyl moiety present advantageously containsfrom 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, moreparticularly from 1 to 4 carbon atoms. Any cycloalkyl moiety present insuch esters advantageously contains from 3 to 6 carbon atoms. Any arylmoiety present in such esters advantageously comprises a phenyl group.Any reference to any of the above compounds also includes a reference toa physiologically acceptable salt thereof.

Preferred derivatives of 1592U89 are the mono-, di-, and tri-phosphateesters of (1R, 4S)-9-[4-(hydroxymethyl)-2-cyclopenten-1-yl]guanine(carbovir).

Examples of physiologically acceptable salts of 1592U89 and theirphysiologically acceptable derivatives include salts derived from anappropriate base, such as an alkali metal (for example, sodium), analkaline earth (for example, magnesium), ammonium and NX₄ ⁺ (wherein Xis C₁₋₄ alkyl). Physiologically acceptable salts of an hydrogen atom oran amino group include salts of organic carboxylic acids such as acetic,lactic, tartaric, malic, isethionic, lactobionic, and succinic acids,organic sulphonic acids, such as methanesulphonic, ethanesulphonic,benzenesulphonic and p-toluenesulphonic acids and inorganic acids, suchas hydrochloric, sulphuric, phosphoric and sulphamic acids.Physiologically acceptable salts of a compound of an hydroxy groupinclude the anion of said compound in combination with a suitable cationsuch as Na⁺, NH₄ ⁺ and NX₄ ⁺ (wherein X is a C₁₋₄ alkyl group).

For therapeutic use, salts of 1592U89 will be physiologicallyacceptable, i.e. they will be salts derived from a physiologicallyacceptable acid or base. However, salts of acids or bases which are notphysiologically acceptable may also find use, for example, in thepreparation or purification of a physiologically acceptable compound.All salts, whether or not derived from a physiologically acceptable acidor base, are within the scope of the present invention.

Preferred salts of 1592U89 are the succinate salt and the hemisulfatesalt.

The formulations according to the invention may be presented in variousforms adapted for direct oral administration including liquid forms, forexample, syrups, suspensions, or solutions. The formulations, accordingto the invention, may include other pharmaceutically acceptable carriersas excipients conventionally used in such formulations.

The compositions of the present invention may be formulated usingmethods and techniques suitable for the compositions' physical andchemical characteristics and that are commonly employed by personsskilled in the art of preparing oral dosage forms (Remington, TheScience and Practice of Pharmacy, 19th ed., 1995).

The compositions according to the invention may be employed in medicaltherapy in combination with other therapeutic agents suitable in thetreatment of HIV infections, such as nucleoside reverse transcriptaseinhibitors for example zidovudine, zalcitabine, didanosine, stavudine,5-chloro-2′,3′-dideoxy-3′-fluorouridine, lamivudine, and(2R,5S)-5-fluoro-1-[2-(hydroxymethyl)1,3-oxathiolan-5-yl]cytosine;non-nucleoside reverse transcriptase inhibitors for example HEPT, TIBOderivatives, atevirdine, L-ofloxacin, L-697,639, L-697-661, nevirapine(BI-RG-587), loviride (α-APA), delavuridine (BHAP), phosphonoformicacid,(−)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one(L-743,726 or DMP-266), and isopropyl(2S)-7-fluoro-3,4-dihydro-2-ethyl-3-oxo-1-(2H)-qinoxalinecarboxylate(HBY 1293); HIV protease inhibitors for example saquinavir, indinavir,ritonavir, nelfinavir, and 141W94; other anti-HIV agents for examplesoluble CD4; immune modulators for example interleukin II,erythropoetin, tucaresol; and interferons for example α-interferon.

According to another aspect, the present invention provides a method forthe treatment of an HIV infection in an infected animal, for example, amammal including a human, which comprises treating said animal with acomposition according to the invention.

Reference herein to treatment extends to prophylaxis as well as thetreatment of established infections, symptoms, and associated clinicalconditions such as AIDS related complex (ARC), Kaposi's sarcoma, andAIDS dementia.

The present invention also provides the use of a composition, ashereinbefore described, in the manufacture of a medicament for thetreatment and/or prophylaxis of HIV infections and associated clinicalconditions hereinbefore described.

In general a suitable dose of 1592U89 for administration to a human fortreatment of an HIV injection may be in the range of 0.1 to 120 mg perkilogram body weight of the recipient per day, preferably in the rangeof 3 to 90 mg per kilogram body weight per day and most preferably inthe range 5 to 60 mg per kilogram body weight per day.

Unless otherwise indicated all weights of active ingredients arecalculated in terms of the drug per se. In the case of a physiologicallyfunctional derivative of 1592U89 or a solvate of any thereof the figureswould be increased proportionately. The desired dose may preferably bepresented as one, two, three, four, five, six or more sub-dosesadministered at appropriate intervals throughout the day. Thesesub-doses may be administered in unit dosage forms, for example,containing from 1 to 1500 mg, preferably from 5 to 1000 mg, mostpreferably from 10 to 700 mg of active ingredient per unit dosage form.Alternatively, if the condition of the recipient so requires, the dosemay be administered as a continuous infusion.

Pharmaceutical compositions according to the present invention maycontain one or more pharmaceutically acceptable carriers or excipientsand optionally other therapeutic agents. The carrier(s) must beacceptable in the sense of being compatible with the other ingredientsof the formula and not deleterious to the recipient thereof.

The present invention may conveniently be presented as a pharmaceuticalcomposition in a unit dosage form. A convenient unit dosage compositioncontains the active ingredients in amounts of from 50 mg to 3 g each,for example, 100 mg to 2 g.

The concentration of 1592U89 hemisulfate salt may be 1-90 mg/ml at a pHrange of about 2.0 to about 4.5.

The unit dosage form may be prepared by any methods well known in theart of pharmacy. Such methods represent a further feature of the presentinvention and include the step of bringing into association the activeingredients with the carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product

Preferred unit dosage compositions are those containing a daily dose ordaily subdose of the active ingredients, as hereinbefore recited, or anappropriate fraction thereof.

Pharmaceutical compositions are often prescribed to the patient in“patient packs” containing the whole course of treatment in a singlepackage, usually a blister pack or a foil pouch. Patient packs have anadvantage over traditional prescriptions, where a pharmacist divides apatient's supply of a pharmaceutical from a bulk supply, in that thepatient always has access to the package insert contained in the patientpack, normally missing in traditional prescriptions. The inclusion of apackage insert has been shown to improve patient compliance with thephysicians instructions and, therefore, lead generally to moresuccessful treatment.

It should be understood that in addition to the ingredients particularlymentioned above the compositions of this invention may include otheragents conventional in the art, for example, those suitable for oraladministration may include such further agents as sweeteners, thickenersand flavoring agents.

1592U89 may be prepared by the method described in European PatentSpecification Number 0434450 or PCT Application No. GB95/00225 which areincorporated herein by reference hereto.

The succinate salt of 1592U89 may be prepared by the method described inPCT Application No. GB95/02014, which is incorporated herein byreference hereto.

The following examples are intended for illustration only and are notintended to limit the scope of the invention in any way.

EXAMPLE 1

Preparation of(1S,4R)-cis-4-[2-Amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt

A stirred mixture of water (25 ml) and isopropanol (IPA) (100 ml) washeated to 45 to 55° C. and(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsuccinate salt (WO 96/06844) (50 g)) was added, and washed in with IPA(12.5 ml). The mixture was heated under reflux for about 0.5 h to give aclear solution and then cooled to 65 to 75° C. and a solution ofconcentrated sulfuric acid (6.07 g) in water (12.5 ml) was added. Amixture of IPA (37.5 ml) and water (12.5 ml) was added and the solutionwas cooled to 45 to 55° C., whereupon a seed of authentic(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt was added. After stirring in this temperature range forabout 1 h to allow crystallisation to become established, further IPA(300 mL) was added, maintaining the temperature of the mixture in therange 45 to 55° C. The suspension was cooled to 0 to 5° C. over about 2h, and the product was filtered, washed with IPA (2×75 ml), and dried invacuo at 40 to 45° C. to give the title compound as a fawn coloredpowder (34.3 g, 90%); m.p. 224-225° C. (decomp.); 1H-NMR (DMSO-d6) δ:10.76(br m, 1, purine NH), 8.53(vbr m, 1, NH), 7.80(s,1,purine CH),6.67(br m, 1, NH2), 6.13(m,1, ═CH), 5.87(m,1,═CH), 5.40(m,1,NCH),3.45(d, J=5.8 HZ,2, OCH2), 2.96(br, m, 1 CH of cyclopropyl), 2.87(m, 1,CH), 2.67-2.57 (M, 1, CH), 1.65-1.55(m, 1, CH), 0.84-0.64(m, 4, 2×CH2 ofcyclopropyl).

EXAMPLE 2

(1S,4R)-cis-4-[2-Amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol(1592U89 Hemisulfate) oral solution

A. Composition A Grade Quantity/ Component (USP/NF) Dosage Unit Function1592U89 Hemisulfate 23.4¹ Active Sorbitol^(2.3) NF 344.4 SweetenerSaccharin Sodium³ USP 0.3 Sweetener Artificial Strawberry 2.0 FlavorFlavor³ Artificial Banana Flavor³ 2.0 Flavor Sodium Citrate USP 10.0 pHAdjustment (Dihydrate) Citric Acid (Anhydrous) USP 7.0 pH AdjustmentMethylparaben NF 1.5 Preservative Propylparaben NF 0.18 PreservativePropylene Glycol USP 50.0 Solubility Enhancer Diluted Hydrochloric NF topH 4.0⁵ pH Adjustment Acid/ Solution of Sodium Hydroxide Purified Water⁴USP to 1.0 mL Vehicle Notes: ¹Hemisulfate salt converted to base usingfactor of 1.17, may be adjusted for purity. ²Sorbitol Solution USP orNoncrystallizing Sorbitol Solution NF may be substituted for SorbitolNF. ³Quantity may vary ± 10% from that listed. ⁴Water for injection USPmay be used in place of Purified Water USP throughout the manufacturingprocess. ⁵The pH may range from 3.8-4.5.

B. Preparation

500 L Batch Size, 20 mg(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol per mL

1. Use of Sorbitol Solution

40% of the Propylene Glycol USP was added to an appropriately-sizedauxiliary vessel. Methylparaben NF and Propylparaben NF was added to thePropylene Glycol USP with mixing, and mixed until dissolved. PurifiedWater USP was dispensed into a stainless steel manufacturing tankequipped with a suitable mixer to approximately 40% of final batchvolume. The appropriate volume of Sorbitol Solution USP was dispensedinto the manufacturing tank. While mixing, the 1592U89 Hemisulfate wasadded and mixed until dissolved. While continuing to mix, theparaben/glycol solution, the remaining Propylene Glycol USP, artificialstrawberry flavor, artificial banana flavor, Saccharin Sodium NF, CitricAcid Anhydrous USP, and Sodium Citrate Dihydrate USP were added andmixed until dissolved. The mixer was turned off and the solution broughtto a volume of 500 L and mixed until a homogeneous solution wasachieved. The solution was sampled and the pH measured. The pH wasadjusted to 3.8-4.5 with NaOHor HCl solution. The final solution wasfiltered through a clarifying filter into an appropriately-sizedreceiving vessel. Clean, compressed, filtered air was blown into bottlesand the bottles were filled with 1592U89 hemisulfate oral solution,capped, and torqued. Alternatively, Water for Injection USP may be usedin place of Purified Water USP throughout the manufacturing procedure.

2. Use of Sorbitol NF

Alternatively, Sorbitol NF may be used in place of Sorbitol SolutionUSP. Purified Water USP was added to a volume of approximately 70% ofthe batch in a stainless steel manufacturing tank equipped with asuitable mixer. While mixing, the Sorbitol NF was mixed until dissolved.While mixing, the 1592U89 Hemisulfate was added and mixed untildissolved. While continuing to mix, the paraben/glycol solution, theremaining Propylene Glycol USP, artificial strawberry flavor, artificialbanana flavor, Saccharin Sodium NF, Citric Acid Anhydrous USP, andSodium Citrate Dihydrate USP were added and mixed until dissolved. Themixer was turned off and the solution brought to a volume of 500 L andmixed until a homogeneous solution was achieved. The solution wassampled and the pH measured. The pH was adjusted to 4.0 with NaOH/HClsolution. The final solution was filtered through a clarifying filterinto an appropriately-sized receiving vessel. Clean, compressed,filtered air was blown into bottles and the bottles were filled with1592U89 hemisulfate oral solution, capped, and torqued.

EXAMPLE 3

(1S,4R)-cis-4-[2-Amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol(1592U89 Hemisulfate) oral solution

A. Composition B Quantity/Dosage Unit Component (mg/mL) 1592U89Hemisulfate 23.4¹ Fructose 200.0 Saccharin Sodium 1.0 Acesulfame K 5.0Artificial Strawberry Flavor 2.0 Artificial Banana Flavor 2.0 SodiumCitrate (Dihydrate) 10.0 Citric Acid (Anhydrous) 7.0 Methylparaben 1.5Propylparaben 0.18 Propylene Glycol 50.0 Diluted Hydrochloric Acidand/or to pH 4.0 solution of Sodium Hydroxide⁴ Purified Water to 1.0 mL¹Hemisulfate salt converted to base using factor of 1.17, may becorrected for purity.

B. Preparation

40% of the Propylene Glycol USP was added to an appropriately-sizedauxiliary vessel. Methylparaben NF and Propylparaben NF was added to thePropylene Glycol USP with mixing, and mixed until dissolved. PurifiedWater USP was added to a volume of approximately 70% of the batch in astainless steel manufacturing tank equipped with a suitable mixer. Whilemixing, the Fructose USP was mixed until dissolved.While mixing, the1592U89 Hemisulfate was added and mixed until dissolved. Whilecontinuing to mix, the paraben/glycol solution, the remaining PropyleneGlycol USP, artificial strawberry flavor, artificial banana flavor,Saccharin Sodium NF, acesulfame, Citric Acid Anhydrous USP, and SodiumCitrate Dihydrate USP were added and mixed until dissolved. The mixerwas turned off and the solution brought o a volume of 500 L and mixeduntil a homogeneous solution was achieved. The solution was sampled andthe pH measured. The pH was adjusted to 3.8-4.5 with NaOHor HClsolution. The final solution was filtered through a clarifying filterinto an appropriately-sized receiving vessel. Clean, compressed,filtered air was blown into bottles and the bottles were filled with1592U89 hemisulfate oral solution, capped, and torqued.

Alternatively, Water for Injection USP may be used in place of PurifiedWater USP throughout the manufacturing procedure.

EXAMPLE 4

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

(1S,4R)-cis-4-[2-amino-6-chloro-9H-purin-9-yl]-2-cyclopentene-1-methanolhydrochloride salt (EP0434450 (80 g)) was heated under reflux inindustrial methylated spirits (IMS, 800 ml) with cyclopropylamine (110ml) for about 5 h. The mixture was cooled to 70 to 75° C. and an aqueoussolution of sodium hydroxide (10M, 55 ml, 2 molar equivalents) was addeddropwise. The resultant suspension was cooled to 20 to 25° C. andfiltered, the collected solids being washed with IMS (2×60 ml). Thecombined filtrates and washings were treated with charcoal (8 g) and thefilter-aid Harborlite J2 (4 g) then heated to 40 to 50° C. After about0.5 h, the mixture was cooled to 15 to 20° C. and the solids wereremoved by filtration, washed with IMS (2×60 ml and 1×80 ml) and thecombined filtrates and washings were concentrated by distillation underreduced pressure to a residual volume of about 240 ml. IMS (560 ml) wasadded and the mixture was concentrated under reduced pressure to aresidual volume of about 240 ml. The dilution and reconcentration wasrepeated and the resultant concentrate was diluted with IMS (240 ml) andheated to obtain a complete solution which was divided into four equalportions.

One portion was concentrated by distillation under reduced pressure to aresidual volume of about 60 ml. Acetone (140 ml) was added and themixture re-concentrated to about 60 ml. This dilution andre-concentration was repeated twice to give a fluid volume of about 80ml. The resultant suspension was cooled to 0 to 5° C. and the productwas filtered, washed with cold (0 to 5° C.) acetone (2×40 ml) and driedin vacuo to give the title compound as an orange solid (16.8 g, 90%);¹H-NMR (D₂O) δ: 7.71(s, 1, purine CH), 6.22(m, 1, ═CH), 5.93(m, 1, ═CH),5.37(m, 1, NCH), 3.61(m, 2, OCH₂), 3.04(br m, 1, CH of cyclopropyl),2.82(br m, 1,CH), 2.80-2.70(m, 1, CH), 1.58-1.50(m, 1, CH), 0.90-0.60(m,4, 2×CH ₂ of cyclopropyl).

EXAMPLE 5

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt

A stirred mixture of water (25 ml) and IPA (100 ml) was heated to 45 to55° C. and(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsuccinate salt (WO96/06844 (50 g)) was added, and washed in with IPA(12.5 ml). The mixture was heated under reflux for about 0.5 h to give aclear solution and then cooled to 65 to 75° C. and a solution ofconcentrated sulfuric acid (6.07 g) in water (12.5 ml) was added. Amixture of IPA (37.5 ml) and water (12.5 ml) was added and the solutionwas cooled to 45 to 55° C., whereupon a seed of authentic(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt was added. After stirring in this temperature range forabout 1 h to allow crystallisation to become established, further IPA(300 ml) was added, maintaining the temperature of the mixture in therange 45 to 55° C. The suspension was cooled to 0 to 5° C. over about 2h, and the product was filtered, washed with IPA (2×75 ml), and dried invacuo at 40 to 45° C. to give the title compound as a fawn colouredpowder (34.3 g, 90%); m.p. 224-225° C. (decomp.); ¹H-NMR (DMSO-d6) δ:10.76(br m, 1, purine NH), 8.53(vbr m, 1, NH), 7.80(s, 1, purine CH),6.67(br m, 1, NH₂), 6.13(m, 1, ═CH), 5.87(m, 1, ═CH), 5.40(m, 1, NCH),3.45(d, J=5.8 Hz, 2, OCH₂), 2.96(br m, 1, CH of cyclopropyl), 2.87(m, 1,CH), 267-2.57(m, 1, CH), 1.65-1.55(m, 1, CH), 0.84-0.64(m, 4, 2×CH ₂ ofcyclopropyl).

EXAMPLE 6

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt

A stirred suspension of(1S,4R-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsuccinate salt (WO96/06844 (1000 g)) in industrial methylated spirit(IMS) (7000 ml) was heated under reflux for about 0.5 h to obtain aclear solution. The solution was cooled to about 70° C. and a solutionof concentrated sulfuric acid (121 g) in IMS (1000 ml) was added. Afterseeding with authentic(1S,4R-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol hemisulfate salt, the mixture was stirred at about 70° C. toallow the product to crystallise. After about 0.5 h, the mixture wascooled to 20 to 30° C. over about 2 h. The mixture was filtered, thecake was washed with IMS (2×2000 ml) and dried in vacuo at 40 to 45° C.to give the title compound as a fawn coloured powder (764 g, 92%),spectra identical to those of the product of Example 5.

EXAMPLE 7

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt

A suspension of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsuccinate salt (10 g) in industrial methylated spirit (IMS) (30 ml) andwater (5 ml) was heated under reflux for about 0.5 h to give a clearsolution. The solution was cooled to 55 to 65° C. and a solution ofconcentrated sulfuric acid (1.21 g) in water (2.5 ml) was added,followed by a mixture of IMS (7.5 ml) and water (2.5 ml). The solutionwas further cooled to 45 to 55° C. and acetone (80 ml) was added overabout 0.25 h to the mixture within this temperature range. The resultantsuspension was cooled to 0 to 5° C. over about 1 h. The product wasfiltered, washed with acetone (2×10 ml) and dried in vacuo at 40 to 45°C. to give the title compound as a fawn coloured powder (6.28 g, 82%)which was spectroscopically identical to the product of Example 5.

EXAMPLE 8

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt

(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol(Intermediate 1) (5.98 g) was suspended in IMS (40 ml) and thesuspension was heated under reflux for about 0.5 h. The mixture wascooled to 70 to 75° C. and a mixture of a solution of concentratedsulfuric acid in IMS (10M, 1.03 ml, 0.5 molar equivalent) and IMS (10ml) was added dropwise. The acid was washed in with further IMS (10 ml)and the resultant suspension was cooled to 0 to 5° C. The product wasisolated by filtration, washed with IMS (2×12 ml) and dried in vacuo at40 to 45° C. to yield the title compound as a pale yellow solid (6.15 g,88%), spectra identical to those of the product of Example 5.

EXAMPLE 9

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt

A further portion of the IMS solution of Intermediate 1 was heated to 75to 80° C. to ensure complete solution. This was cooled to 70 to 75° C.and a solution of concentrated sulfuric acid (3.90 g) in IMS (30 ml) wasadded dropwise, to give an orange coloured suspension. The mixture wascooled to 0 to 5° C. over about 2 h and the product was filtered, washedwith IMS (2×40 ml) and dried in vacuo at 40 to 45° C. to give the titlecompound as a yellow/orange solid (17.7 g, 76%), spectra identical tothose of the product of Example 5.

Of this product, 5.0 g was suspended in a mixture of isopropanol (IPA)(40 ml) and water (10 ml) and heated under reflux for about 0.5 h andthen allowed to cool to 55 to 60° C., whereupon seeds of authentic(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt were added. The suspension was cooled further to 0 to5° C. and the temperature was maintained for about 1 h. The solid wasfiltered, washed with IPA (2×5 ml) and dried in vacuo at 40 to 45° C. toyield the title compound as a buff coloured powder (4.4 g, 88%), spectraidentical to those of the product of Example 5.

EXAMPLE 10

(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolbenzoate salt

(1S,4R)-cis-4-[2-amino-6-chloro-9H-purin-9-yl]-2-cyclopentene-1-methanolhydrochloride salt EP0434450 (70 g) was heated under reflux in IMS (700ml) with cyclopropylamine (94.5 ml) for about 4 h. The solution wascooled to 45 to 50° C. and treated with filter-aid Harborlite J2 (3.5 g)and charcoal (7 g). After about 0.5 h, the mixture was cooled to 20 to25° C. and filtered. The solids were washed with IMS (2×140 ml) and thecombined filtrates and washings were concentrated by distillation underreduced pressure to a volume of about 210 ml. After dilution with IMS,(490 ml) the solution was re-concentrated to about 210 ml. The dilutionand reconcentration was repeated once and the final concentrate wasdivided into seven equal portions. One portion was diluted with IMS (80ml) and warmed until a complete solution was obtained. Benzoic acid(4.85 g) was added as a single portion and the mixture was heated at 70to 75° C. to give a complete solution, which was then allowed to coolslowly. At 40 to 45° C. the mixture was seeded with authentic(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolbenzoate salt and the mixture was further cooled to 0 to 5° C. The solidwas filtered, washed with IMS (2×20 ml) and dried in vacuo at 40 to 45°C. to give the title compound as a white solid (8.7 g, 64%), mpt:156-157° C.; ¹H-NMR (DMSO-d₆) δ: 7.95(m, 2, benzoate CH), 7.63(m, 1,benzoate CH), 7.61(s, 1, purine CH), 7.50(m, 2, benzoate CH), 7.28(br m,1, NH), 6.11(m, 1, ═CH), 5.86(m, 1, ═CH), 5.81 (br m, 1, OH), 5.39(m, 1,NCH), 3.45(d, J=6.0 Hz, 2, OCH₂), 3.04(br m, 1,CH of cyclopropyl),2.87(br m, 1, CH), 2.65-2.55(m, 1, CH), 1.63-1.53(m, 1, CH),0.70-0.54(m, 4, 2×CH ₂ of cyclopropyl).

EXAMPLE 11

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt

A suspension of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolbenzoate salt (5 g) in IPA (25 ml) was warmed to 60 to 65° C. A solutionof concentrated sulfuric acid (0.64 g) in water (1.25 ml) was added andthe resultant cloudy suspension was warmed to 70 to 75° C. The mixturewas cooled to 20 to 25° C. and filtered. The solid was washed with IPA(2×10 ml) and dried in vacuo at 40 to 45° C. to give the title compoundas a white solid (3.57 g, 87%), spectra identical to those of theproduct of Example 5.

EXAMPLE 12

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolglutarate salt

(1S,4R)-cis-4-[2-amino-6-chloro-9H-purin-9-yl]-2-cyclopentene-1-methanolhydrochloride salt (EP0434450) (80 g) was heated under reflux in IMS(800 ml) with cyclopropylamine (108 ml) for about 3.5 h. The solutionwas cooled to 45 to 50° C. and treated with charcoal (8 g) andfilter-aid Harborlite J2 (4 g). After about 1 h, the mixture was cooledto 20 to 25° C. and filtered. The solids were washed with IMS (2×160 ml)and the combined filtrates and washings were concentrated bydistillation under reduced pressure to about 240 ml. The mixture wasdiluted with IMS (560 ml) and re-concentrated to about 240 ml. Theprocess of dilution and re-concentration was repeated twice more. Thefinal concentrate was divided into four equal portions. One portion washeated to 70 to 75° C. to give a solution. To this was added a solutionof glutaric acid (8.75 g) in water (144 ml) which had been pre-heated to70 to 75° C. The mixture was cooled to 60 to 65° C. and seeded withauthentic(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolglutarate salt. The mixture was cooled to 0 to 5° C. and filtered. Theproduct was washed with a mixture of water and IMS (4:1, 2×36 ml) anddried in vacuo at 40 to 45° C. to furnish the title compound as a lightbrown solid (19.9 g, 80%); mp. 184-188° C.; ¹H-NMR (DMSO-d₆) δ: 7.60(s,1, purine CH), 7.27(br m, 1, NH), 6.10(m, 1, ═CH), 5.86(m, 1, ═CH),5.82(br m, 1, OH), 5.39(m, 1, NCH), 3.44(d, J=5.9 Hz, 2, OCH2), 3.04(brm, 1, CH of cyclopropyl), 2.87(br m, 1, CH), 2.65-2.55(m, 1, CH),2.24(t, J=7.2 Hz, 4, glutarate 2×CH ₂), 1.70(m, J=7.2 Hz, 2, glutarateCH₂), 1.62-1.54(m, 1, CH), 0.68-0.54(m, 4, 2×CH ₂ of cyclopropyl).

EXAMPLE 13

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt

A suspension of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolglutarate salt (10 g) in IPA (50 ml) was heated to 60 to 65° C. and asolution of concentrated sulfuric acid (1.18 g) in water (2.5 ml) wasadded. The resultant suspension was warmed further to 70 to 75° C. andthen cooled to 20 to 25° C. The product was filtered, washed with IPA(2×20 ml) and dried in vacuo at 40 to 45° C. to give the title compoundas a light brown solid (6.78 g, 85%), spectra identical to those of theproduct of Example 5.

EXAMPLE 14

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt from the succinate salt in the presence of itsenantiomer

A mixture of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,succinate salt and its enantiomer (134 g) having an enantiomeric ratioof 97.5:2.5 as shown by chiral HPLC (eluant (1.0 v/v acetonitrile inaqueous 0.05M potassium phosphate buffer, pH 6.5; column ChromTechChiral-AGP, 100×4.0 mm; flow 1.0 ml/min;detection at 220 nm) wassuspended in isopropanol (IPA) (302 ml) and water (67 ml) and heated toreflux to give a clear solution. The solution was cooled to 75 to 80° C.and a solution of concentrated sulfuric acid (16.26 g) in water (33.5ml) was added, and the solution was clarified by a hot filtration,following through the filter with a mixture of IPA and water (3:1, 134ml). The filtrates and washings were cooled to 45 to 50° C. and seededwithauthentic(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,hemisulfate salt. Further IPA (804 ml) was added in this temperaturerange and the resultant suspension was cooled to 0 to 5° C. Thesuspension was filtered and the product was washed with IPA (2×200 ml)and dried in vacuo at 40 to 45° C. to give the title compound as a whitecrystalline solid (75 g, 68%).

Analysis of the product by chiral HPLC (conditions as above) showed theratio of enantiomers to be 99.2:0.8.

A range of similar experiments was carried out on 8 g scale usingdifferent ratios of enantiomers of the input succinate salt with thesame experimental protocol. The results are summarised below in tabularform:

RATIO OF ENANTIOMERS OF RATIO OF ENANTIOMERS OF INPUT SUCCINATE SALTPRODUCT HEMISULFATE SALT 99.5:0.5 99.87:0.1 99.0:1.0 99.72:0.3 98.0:2.099.47:0.5 96.0:4.0 98.97:1.0

EXAMPLE 15

Preparation of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhemisulfate salt from the glutarate salt in the presence of itsenantiomer

A mixture of(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,glutarate salt and its enantiomer (100 g) having an enantiomeric ratioof 98.6:1.4 as shown by chiral HPLC (conditions as above in Example14)was suspended in isopropanol (IPA) (400 ml) and water (100 ml) andheated to reflux to give a clear solution. The solution was cooled to 70to 75° C. and a solution of concentrated sulfuric acid (12.01 g) inwater (25 ml) was added, followed by a mixture of IPA and water (4:1,100 ml) and then by IPA (100 ml). The solution was cooled to 50 to 55°C. and seeded with authentic(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,hemisulfate salt. Further IPA (800 ml) was added in this temperaturerange and the resultant suspension was cooled to 0 to 5° C. Thesuspension was filtered and the product was washed with IPA (2×200 ml)and dried in vacuo at 40 to 45° C. to give the title compound as a whitecrystalline solid (72 g, 90%).

Analysis of the product by chiral HPLC (conditions as above inExample14) showed the ratio of enantiomers to be 99.6:0.4.

The application of which this description and claims form part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofproduct, composition, process or use claims and may include, by way ofexample and without limitation, one or more of the following claims.

What is claimed is:
 1. A pharmaceutical composition for oraladministration comprising(1S,4R)-cis-4-[(2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,or a pharmaceutically acceptable derivative thereof, together withcitrate and at least one sweetener selected from sorbitol and saccharin,at a pH range of 2.0 to 4.5.
 2. A pharmaceutical composition for oraladministration comprising(1S,4R)-cis-4-[(2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,or a pharmaceutically acceptable derivative thereof, together withcitrate and at least one sweetener selected from sorbitol and saccharin,at a pH range of 6.6 to 7.5.
 3. A pharmaceutical composition as claimedin claim 1 wherein the sweetener is saccharin and the compositionfurther comprises fructose and acesulfame.
 4. A pharmaceuticalcomposition as claimed in claim 2 wherein the sweetener is saccharin andthe composition further comprises fructose and acesulfame.
 5. Apharmaceutical composition as claimed in claim 1 wherein thepharmaceutically acceptable derivative of(1S,4R)-cis-4-[(2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolis the hemisulfate salt.
 6. A process for formulating(1S,4R)-cis-4-[(2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolfor oral administration comprising the step of bringing(1S,4R)-cis-4-[(2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,or a pharmaceutically acceptable derivative thereof, into associationwith citrate and a sweetening agent which does not contain a carboxylgroup.
 7. A process as claimed in claim 6 wherein the(1S,4R)-cis-4-[(2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolis in the form of its hemisulfate salt.
 8. A pharmaceutical compositionfor oral administration comprising(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,or a pharmaceutically acceptable derivative thereof, citrate, and asweetening agent compatible with said(1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,or pharmaceutically acceptable derivative thereof.
 9. A pharmaceuticalcomposition as claimed in claim 8 at a pH range of 2.0 to 4.5.
 10. Apharmaceutical composition according to claim 9 wherein the pH range is3.8.
 11. A pharmaceutical composition according to claim 10 wherein thepH is 4.1.
 12. A pharmaceutical composition as claimed in claim 8wherein the sweetening agent is selected from the group consisting ofsorbitol, saccharin, acesulfame, fructose, sucralose, and aspartame. 13.A pharmaceutical composition according to claim 8 wherein the citrateion concentration is in the range of 0.01 M to 0.1 M.
 14. Apharmaceutical composition according to claim 1 in the form of asolution.